HbS interacts with the TLR4/MD-2 complex and is responsible for TLR4-mediated monocyte activation, independently of LPS, ROS, NO, or heme. (A) SPR analysis of the binding between HbS (50 nM), HbA (50 nM), or BSA (200 nM) and the TLR4/MD-2 complex. (B-C) Levels of TNF-α (B) and IL-6 (C) in the cell culture supernatant of human monocytes from healthy blood donors (n = 9), in culture with HbA (20 μM), HbS (20 μM), HbS (20 μM) pretreated with polymyxin B (100 μg/mL) for 2 hours, HbS (20 μM) pretreated with proteinase K (5 mg/mL) at 37°C for 3 hours, HbS (20 μM) with TAK-242 (1 μM) added to culture medium 1 hour before HbS, or control, after 18 hours (n = 3 to 9 per group). (D) NF-κB activation level of THP1-Blue cells in culture with HbA (20 μM), HbS (20 μM), NAC (50 μM), HbA (20 μM) with NAC (50 μM), HbS (20 μM) with NAC (50 μM), or control, after 18 hours (n = 3 per group). (E-F) IL-6 levels in the cell culture supernatant of human monocytes from healthy blood donors (n = 3), in culture with HbS (20 μM), HbS (20 μM) with catalase (500 U/mL), SOD (100 U/mL), L-NAME (5 mM), ODQ (100 μM), NONOate (1 mM), hemopexin (100 μg/mL), or control, after 18 hours (n = 3 per group). ∗∗∗P < .001 by one-way ANOVA. NO, nitric oxide; ROS, reactive oxygen species.